The second complete mitochondrial genome of Capillidium rhysosporum within the family Capillidiaceae, Entomophthorales

Abstract The complete mitochondrial genome of the entomophthoroid fungus Capillidium rhysosporum (strain no.: ATCC 12588) was sequenced using next-generation sequencing technology. The assembled circular genome has a length of 46,756 base pairs with a GC content of 27.06%. Gene prediction identified 15 core protein-coding genes (PCGs), two rRNA genes, and 27 tRNA genes. Phylogenetic analysis confirmed that C. rhysosporum belongs to the Zoopagomycota clade and is closely related to C. heterosporum. This study presents the second complete mitochondrial genome within the family Capillidiaceae, contributing to the mitochondrial DNA database of entomophthoroid fungi.


Introduction
The genus Capillidium B. Huang & Y. Nie 2020 was recently distinguished from Conidiobolus (Brefeld 1884) based on its unique characteristic of capilliconidia arising from conidia (Nie et al. 2020).Subsequently, it was reclassified into the family Capillidiaceae Y. Nie, Stajich & K.T. Hodge 2022 based on molecular data, ancestral lifestyles, and morphological features (Gryganskyi et al. 2022).With the addition of two more species in 2022, there are now 10 known species in this genus (Nie et al. 2022).
In a previous study, we reported the mitochondrial genome of Capillidium heterosporum (Drechsler) B. Huang & Y. Nie 2020 as the first complete mitochondrial genome in the family of Capillidiaceae.Comparative analysis revealed it to have the second-highest number of introns among 22 basal fungi (Nie et al. 2019).Here, we present the sequencing and analysis of the second complete mitochondrial genome of Capillidiaceae, providing insights into phylogenetic relationships and evolutionary histories among entomophthoroid fungi.

Materials and methods
The ex-type strain ATCC 12588 of C. rhysosporum was obtained from the American Type Culture Collection (Manassas, VA), and this species was known for its rough zygospores, and was isolated from decaying plant debris near Laplace (38 � 32 0 N, 76 � 59 0 W), Louisiana, United States in 1952 (the original descriptive image were shown in Figure 1).Fungal cultures were incubated on PDA for seven days at 21 � C. Total genomic DNA was extracted from fresh fungal mycelia using a modified CTAB method (Watanabe et al. 2010).Duplicate specimens and genomic DNA were deposited at the Research Center for Entomogenous Fungi (RCEF), Anhui Agricultural University (www.ahau.edu.cn,Professor Bo Huang, Email: bhuang@ahau.edu.cn),Hefei, Anhui, China under the voucher number RCEF 20081012.The constructed paired-end libraries with 300 bp inserts was used for Illumina HiSeq X-ten sequencing (NextOmics Biosciences, Co., Ltd., Wuhan, China) following the manufacturer's instructions (Bioo Scientific, Austin, TX, AIR TM Paired-End DNA Sequencing Kit).Quality assessment was performed to obtain clean reads from the raw sequencing data by FastQC 0.11.8 (Andrews 2010).The sequencing depth and coverage map were provided in the supplementary materials (Supplementary Figure S1).The mitogenome was assembled using Norgal 1.0 (Al-Nakeeb et al. 2017), and gene annotation was conducted as previously described (Zhang et al. 2017;Li et al. 2021), with tRNA genes identified using tRNAscan-SE v1.3.1 (Lowe and Eddy 1997).Circular mitogenome maps were drawn with PMGmap (Zhang et al. 2023, http://www.1kmpg.cn/pmgmap).CPGView (http://www.1kmpg.cn/cpgview/)was utilized to illustrate the structures of intron-containing genes as shown in Supplementary Figure S2.

Discussion and conclusions
In this article, we reported another complete mitogenome of the genus Capillidium with 46,756 bp in total length.The number and length of fungal mitogenomic introns have an implication for inferring the fungal mitogenome size (Friedrich et al. 2012;Torriani et al. 2014), and we noted that the number of introns ( 27) was fewer than that of C. heterosporum (30).The rps3 gene encodes protein component of the ribosome for protein translation (Bullerwell et al. 2000), which was annotated in the mitogenome of C. rhysosporum.As a note, the mitogenome of C. rhysosporum is the second complete mitogenome in the family Capillidiaceae, which enhances our understanding of the evolution of this fungal group.
In summary, this study presents another complete mitochondrial genome within the family Capillidiaceae.We also conducted the phylogenetic analysis with other related entomophthoroid fungi based on the sequences of 14 protein coding genes, which reveal a close relationship with C. heterosporum.The comprehensive analysis of its mitochondrial genome contributes valuable insights into the phylogenetic relationships and evolutionary history of entomophthoroid fungi.

Figure 2 .
Figure 2. Circular complete mitogenome map of Capillidium rhysosporum genes is represented by different colored blocks.Genes outside and inside the outermost black ring line are transcribed in a clockwise and counter-clockwise direction, respectively.The inner circles represent the genome scale, GC content and distributions of short tandem repeats, long tandem repeats and the dispersed repeats, respectively.The colored parabola in the center circle represents the dispersed repeats.